Our group has been studying the regulation of class I major histocompatibility complex (MHC) receptors in mouse (Ly49) and man (KIR) for many years, and we are pioneers in the identification of molecular mechanisms underlying the variegated expression of these receptors by NK cells. We have discovered many instances of genetic variation in KIR and Ly49 genes that can change the responsiveness of NK cells to MHC on target cells. However, our long-standing collaboration with the Carrington lab in the Cancer and Inflammation Program (CIP), has stimulated our interest in studying the expression of the KIR ligands, and the HLA-C gene in particular. Dr. Carrington's lab has demonstrated significant allelic variation in HLA-C expression, suggesting that both the receptors and ligands are involved in an evolutionary tuning process to produce optimal NK:target cell interactions. We have therefore conducted an extensive characterization of polymorphisms in HLA-C regulatory elements. In the past year, significant progress has been made in the investigation of the transcriptional control of HLA-C expression -a major ligand for the KIR family of human class I MHC receptors expressed by natural killer (NK) cells. We have identified and characterized a novel distal NK cell-specific HLA-C promoter. This discovery has important implications for the study of NK cell education by self MHC molecules. NK cell education has been a major focus in the field, and recent data has suggested a role for MHC expression by the NK cell itself in this process. Our work has defined a novel paradigm for cell-intrinsic regulation of NK cells by HLA-C, and we have discovered a complex system of tissue-specific alternative mRNA splicing that controls HLA-C expression by NK cells in different tissues. Furthermore, we have identified specific HLA-C alleles that have a single base pair polymorphism that inactivates the NK-specific promoter, resulting in reduced HLA-C expression by NK cells. We have found that this polymorphism has a significant effect on NK cell activity, and we are in the process of determining if hematopoietic stem cells donors that have low HLA-C expression on NK cells are associated with a distinct clinical outcome. This study is a component of our participation in a program project grant led by Dr. Jeffery Miller at the University of Minnesota. In addition, we have conducted a thorough analysis of allelic variation of transcription factor binding sites in the HLA-C gene, and identified multiple polymorphisms that account for the variation of HLA-C expression levels in peripheral blood lymphocytes that has been observed by the Carrington lab. Our detailed study of HLA-C has also revealed the presence of trophoblast-specific elements in the HLA-C gene that are not present in HLA-A or HLA-B genes, providing an explanation for the specific expression of HLA-C by trophoblasts. Future plans include: investigating the mechanism of NK cell-intrinsic tuning by HLA-C; using CRISPR technology to confirm functional role of the multiple KIR and HLA-C elements identified; studying clinical correlations of polymorphisms in the KIR and HLA genes.